Download Mechanisms of cell positioning during C. elegans gastrulation

307
Development 130, 307-320
© 2003 The Company of Biologists Ltd
doi:10.1242/dev.00211
Mechanisms of cell positioning during C. elegans gastrulation
Jen-Yi Lee and Bob Goldstein
Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA
Accepted 17 October 2002
SUMMARY
Cell rearrangements are crucial during development. In this
study, we use C. elegans gastrulation as a simple model to
investigate the mechanisms of cell positioning. During C.
elegans gastrulation, two endodermal precursor cells move
from the ventral surface to the center of the embryo, leaving
a gap between these ingressing cells and the eggshell. Six
neighboring cells converge under the endodermal
precursors, filling this gap. Using an in vitro system, we
observed that these movements occurred consistently in the
absence of the eggshell and the vitelline envelope. We found
that movement of the neighbors towards each other is not
dependent on chemotactic signaling between these cells. We
further found that C. elegans gastrulation requires intact
microfilaments, but not microtubules. The primary
mechanism of microfilament-based motility does not appear
to be through protrusive structures, such as lamellipodia or
filopodia. Instead, our results suggest an alternative
mechanism. We found that myosin activity is required for
gastrulation, that the apical sides of the ingressing cells
contract, and that the ingressing cells determine the
direction of movement of their neighboring cells. Based on
these results, we propose that ingression is driven by an
actomyosin-based contraction of the apical side of the
ingressing cells, which pulls neighboring cells underneath.
We conclude that apical constriction can function to
position blastomeres in early embryos, even before
anchoring junctions form between cells.
INTRODUCTION
C. elegans development follows a precise program of cell
divisions (Sulston et al., 1983). The one-cell embryo divides
into the anterior cell AB, which gives rise to most of the
nervous system and epidermis, while the posterior cell, P1,
produces the endoderm, germline and most of the mesoderm
(Fig. 1A). Among the descendents of P1 are several important
players in gastrulation: the endoderm precursors Ea and Ep,
and their neighbors, the germline founder cell P4, and the
granddaughters of MS, which produce primarily mesoderm
(we use ‘MSxx’ to indicate any one granddaughter of MS).
Gastrulation begins with the ingression of Ea and Ep towards
the center of the embryo, leaving a space between the cells and
the eggshell known as the ventral cleft. Meanwhile, MSxx and
P4 move towards each other and fill the ventral cleft (Fig. 1B,C)
(Sulston et al., 1983). In the experiments described in this
paper, we analyzed the displacement of the Ea and Ep cells
relative to their immediate neighbors. For convenience, we
refer to these movements alone as gastrulation, although these
initial movements are followed by internalization of almost
half of the cells in the developing embryo to complete the
entire process of gastrulation (Sulston et al., 1983; Nance and
Priess, 2002).
A major advantage of using C. elegans embryos to study cell
rearrangement is the potential to combine the powerful tools
of direct cell manipulation, cell biology and genetics. An
in vitro culture system exists whereby the eggshell and
vitelline envelope can be removed (which we refer to as
Cell rearrangement is essential in development. Although
morphogenesis involving epithelial sheets has been a topic of
extensive investigation (reviewed by Jacinto et al., 2001), the
mechanisms that govern the movement of individual cells
during development are less well characterized. Individual cell
migrations, such as those exhibited by sea urchin primary
mesenchyme cells during skeletogenesis (Ettensohn, 1991) and
by ascidian notochord cells during notochord formation
(Munro and Odell, 2002), play an important role in early
embryos.
One of the key cell rearrangements during development
is gastrulation, the process by which metazoan embryos
internalize their future endoderm, resulting in proper
positioning of the three germ layers – the endoderm, the
mesoderm and the ectoderm. The mechanisms of gastrulation
differ between animals, ranging from the simple to the
complex. For example, in the nematode Caenorhabditis
elegans, embryos internalize their endoderm by the ingression
of only two cells (Sulston et al., 1983), whereas, in chick
embryos, the process is complex, requiring the coordinated
movement of entire tissues immediately followed by
neurulation (Schoenwolf, 1991). Owing to its simplicity, the
gastrulation process in C. elegans is particularly well suited to
studying individual cell movement and positioning during
development.
Movies available on-line
Key words: Cell migration, In vitro system, Gastrulation,
Morphogenesis, Polarity, Actomyosin cytoskeleton, C. elegans
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J.-Y. Lee and B. Goldstein
devitellinization), and the resulting naked embryos can be
cultured in embryonic growth medium (Edgar, 1995). This in
vitro system was previously used to dissect cell-cell signaling
and cell fate determination (Goldstein, 1992; Shelton and
Bowerman, 1996; Lin et al., 1998; Berkowitz and Strome,
2000), and is thus well suited to study many aspects of
embryogenesis. Use of in vitro models in other animal systems,
such as Keller explants of Xenopus embryos, has allowed
detailed examination of morphogenetic movements at the
cellular level (Wilson and Keller, 1991).
Despite the numerous advantages of using C. elegans to
study morphogenesis, the mechanisms that underlie C. elegans
gastrulation are unknown. The success or failure of gastrulation
is often noted in characterizing mutants, but few studies
have actually addressed the mechanistic requirements for
gastrulation. Proper endoderm fate appears to be required for
gastrulation, as mutations that prevent endoderm specification
also result in a gastrulation-defective phenotype (reviewed by
Maduro and Rothman, 2002). In addition, proper cell cycle
length may also be essential, as early Ea/Ep division appears
to prevent gastrulation (Schierenberg et al., 1980; Knight and
Wood, 1998). Other factors, such as the formation of small
blastocoel-like spaces, an apical-basal polarized distribution of
PAR proteins, and an accumulation of myosin at the ventral
sides of Ea and Ep might play roles in gastrulation (Nance and
Priess, 2002), but their functional requirements have not been
tested. Therefore, it is currently unclear what mechanisms
control this important event.
In this study, we first asked if we could extend the C. elegans
in vitro system to study morphogenetic movements during
gastrulation. We also addressed the following questions. What
are the mechanisms of cell movement? Is chemotaxis
involved? What is the role of the cytoskeleton? What
determines the direction of cell movement?
MATERIALS AND METHODS
Strains and worm maintenance
Nematodes were cultured and handled as described (Brenner, 1974).
Unless indicated, experiments were performed with the wild-type N2
(Bristol) strain. The following mutant and reporter strains were used:
EU855 mom-2(or309) V/nT1[unc-?(n754) let-?](IV;V); BW1943
gad-1(ct226) dpy-11(e224) (V); TH2, an α-tubulin::GFP fusion
construct driven by the pie-1 promoter (Oegema et al., 2001); WH212
unc-119(ed3); ojls4[actin::YFP unc-119(+)]; and KK871 PAR2::GFP. Strains were maintained at 20°C, except for the gad-1 strain,
which was maintained at 16°C, then shifted overnight to the restrictive
temperature of 25°C to obtain affectd embryos, and the transgenic
strains, which were maintained at 25°C. Filming was performed at 2023°C.
Devitellinization and blastomere culture/isolation
Devitellinization and blastomere culture techniques were performed
essentially as described (Edgar, 1995; Goldstein, 1992), with the
following exceptions: 10% hypochlorite (Sigma) was used; the
enzyme solution consisted of 3 units/ml chitinase (Sigma), 6.6 mg/ml
chymotrypsin (Sigma) and 0.1% penicillin-streptomycin (Gibco); and
the Edgar’s Growth Medium (EGM, also called Embryonic Growth
Medium) (Edgar, 1995) did not contain heavy metals. In cases where
a mouth pipette and needle were insufficient for blastomere
separation, a micropipette was pulled by hand over a flame and the
uncut tip was laid down between blastomeres to separate them. In
most experiments, embryos were checked the next day for the
presence of birefringent gut granules, an indicator of proper cell fate
specification (Babu, 1974; Laufer, 1980). A small percentage of
embryos did not make gut granules (~3% of all experiments), and
these were not included in data analyses.
4-D videomicroscopy
Embryos with eggshells were placed in egg buffer on a 0.1% poly-Llysine (Sigma) coated coverslip, inverted over a 3% agar pad, and
sealed with petroleum jelly. Devitellinized embryos and isolated
blastomeres were placed in 15 µl EGM on a coverslip with clay feet
at its corners and sealed with petroleum jelly. Images were acquired
on a C2400-07 Hamamatsu Newvicon video camera (Hamamatsu
Photonics) mounted on a Nikon Eclipse 800 microscope (Nikon
Instrument Group). Time-lapse images were acquired at 1 µm sections
every 30 seconds using 4D Grabber, and subsequently analyzed with
4D Viewer (Integrated Microscopy Resource, University of
Wisconsin, Madison).
Quantification of cell movements
For timing measurements, the beginning of gastrulation movement
was chosen by the frame in each movie where P4 and MSxx began to
move towards each other. Because there is a slight variation in cell
cycle length from embryo to embryo, cell cycle lengths were
normalized: the beginning of movement was expressed as a fraction
of the length of each embryo’s total Ea/Ep cell cycle. For angle
quantification measurements, two different approaches were used
because of a difference between wild-type and mutant embryos (see
Results). In all cases, the angles were measured using a protractor,
with the line joining the centers of the Ea and Ep cell nuclei as the
(0°) reference point. A measurement was taken at the indicated initial
time point, and that value was subtracted from the measurement at the
final time point, to give the total movement. Means and standard
deviations were calculated as described for circular distributions (Zar,
1999). Watson-Williams tests revealed significant differences between
the four embryo types for the three approaches (see Table 1). Multiple
comparisons were made using pair-wise Watson-Williams tests. This
procedure is analogous to using an ANOVA to determine the main
effect of embryo type followed by pair-wise t-tests for multiple means
comparisons between the embryo types. (This type of procedure
produces a real risk of type 1 errors but could not be avoided because
post hoc means comparison tests for circular data have not been
developed.) It is likely that our angle measurements of P1 isolates are
underestimates, as we measured movements in one plane of a
multiplane movie, and cells sometimes moved partially along the zaxis (in or out of the x, y plane observed). For measurements of apical
surface/length, lengths from the apical border of Ea/MSx(x) to Ep/P4
were measured from time-lapse images using Metamorph software,
where the measuring tool was calibrated to micrometers. Two
timepoints were taken: the first was at P4 birth and the second was
taken 20 minutes later. Three independent measurements were taken
from each embryo at each time point, and were then averaged together
to obtain a representative number for each embryo. Six embryos from
each treatment were analyzed.
Laser permeabilization experiments
Embryos were dissected from hermaphrodites, transferred to washed,
0.1% poly-L-lysine coated coverslips, coated with Nile Blue or
Trypan Blue (Sigma), and washed in egg buffer or EGM. After
mounting, the coverslip was sealed on two opposing edges with
petroleum jelly, allowing for transfer of liquids from one side of the
coverslip to the other. Embryos were permeabilized by irradiating the
Nile Blue or Trypan Blue particles with a few low-energy laser pulses
using a VSL-337 nitrogen laser (2 mW, Laser Science) mounted on a
Nikon Eclipse 800 microscope. In experiments with SynaptoRed
(Novagen), also known as FM4-64, 10 µg/ml SynaptoRed in EGM
was washed in and the coverslip subsequently sealed. Confocal
Cell positioning in C. elegans
imaging was performed at three minute intervals with a Zeiss LSM
410 microscope with an argon krypton laser. Embryos were imaged
live. Cytoskeletal inhibitors were introduced at the desired stages by
placing 10 µl at one end of the unsealed coverslip and using a
Kimwipe at the other to wick up the excess liquid. This was repeated
three times to ensure delivery of the drug. In some experiments, the
same procedure was repeated with egg buffer or EGM in order to wash
the drug out of the solution. The following drugs were used: 5 µg/ml
cytochalasin D, 10 µM latrunculin B, 50 µM taxol and 10 µM
nocodazole. All drugs were diluted in DMSO and used at a final
concentration of 1% DMSO in either egg buffer or EGM. To calculate
rate of ingression, kymographs were made using NIH Image v. 1.62
software. A line was drawn through the Ep nucleus, and this line
follows the same slice for the entire duration of the film. The result
is a distance over time graph, measuring the rate of Ep nucleus
ingression. For the kinase inhibitors ML-7, ML-9 and H-7
(Calbiochem), the drugs were not washed in, but already diluted in
EGM at the concentrations noted in the text and in Fig. 7.
Scanning electron microscopy
Blastomeres were cultured and allowed to develop to gastrulation
stages. The cells were washed twice with simplified culture medium
(SCM) (Goldstein, 1995). The embryos were then fixed, post-fixed
and washed as previously described (Priess and Hirsh, 1986). Cells
were placed on a 0.1% poly-L-lysine coated coverslip, and
subsequently dehydrated through a series of graded ethanol washes
(30%, 50%, 70%, 100%, 100%). Critical-point drying was performed
using CO2 as the transition solvent, and the coverslip was mounted
on an aluminum stub and sputter coated to a thickness of 15 nm with
60:40 Au:Pd alloy on a Hummer X Sputtering System (Anatech).
Samples were observed at an accelerating voltage of 20 kV and
photographed using a Cambridge S200 SEM. Images were acquired
using NIH Image software and processed with Adobe Photoshop
software.
Immunofluorescence
Blastomeres were cultured and allowed to develop to gastrulation
stages. They were then washed twice with SCM, fixed with 4%
paraformaldehyde (Electron Microscopy Sciences), and washed three
times with PBT (phosphate-buffered saline with 1% Tween-20). For
phalloidin, embryos were extracted with sonicated 0.5% Triton-X-100
for 5 minutes immediately following fixation. The following
concentrations were used: 1:50 Alexa Fluor 488 Phalloidin (F-actin,
Molecular Probes) and 1:500 mouse α-phosphotyrosine (P-Tyr-102,
Cell Signaling Technology). Alexa Fluor 488-conjugated goat antimouse secondary antibody was used at 1:1000 (Molecular Probes).
Confocal images were obtained as described above. Epifluorescence
imaging was attained with a Hamamatsu Orca II (model C4742-98)
charge-coupled device camera mounted on a Nikon Eclipse E600FN.
Adobe Photoshop software was used to process the images for
publication.
Fluorescent microsphere experiments
For each experiment, 0.2 µm yellow-green fluorescent, carboxylatemodified latex microspheres (Molecular Probes) were freshly diluted
1:4 in SCM, sonicated for 1 minute and added to P1 isolates in EGM.
The microspheres and P1 isolates were quickly mixed together using
a mouth pipette for three minutes, after which the P1 isolates were
transferred through two washes of EGM. The microspheres attach
non-specifically to protein surfaces (Wang et al., 1994). The isolates
were placed in 2 µl of EGM in one well of an eight-well slide (ICN),
with the three surrounding wells also containing 2 µl of EGM. A
coverslip was gently placed over the four wells, and sealed with valap
(1:1:1 vaseline, lanolin and paraffin). Simultaneous DIC and GFP
time-lapse images were acquired every 30 seconds at three planes, 11.5 µm apart, using a Hamamatsu Orca II (model C4742-98) chargecoupled device camera mounted on a Nikon Eclipse E600FN. The
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Metamorph software package (Universal Imaging) was used for
microscope automation, image acquisition and image analysis. DIC
and GFP images were combined into one image for analysis.
Microsphere movements were traced by hand onto transparencies,
from the beginning to the end of gastrulation movements, or until the
microspheres went out of plane. As both the microspheres and the
cells were moving at this time, the tracing template position was
adjusted at each time interval along with cell movement, to make it
possible to discern microspheres that were moving faster relative to
cell boundaries from those remaining stationary on a moving cell.
Kymographs of bead movement on Ea and Ep were made using
Metamorph software to confirm tracing data. Tracings were scanned
into the computer as TIFF images and then re-drawn using Canvas
software. For vector addition, each cell was divided up into quadrants,
and vectors were added if quadrants contained more than three
vectors. Microspheres along cell boundaries or those that did not move
were not included in the calculations. All calculations were performed
by decomposing the vectors into x and y components. To obtain the
average velocities for the set of microspheres from each quadrant, we
first took the average of the x and y components separately, then
extrapolated the total distance the microsphere would be expected to
move by multiplying the distance from the added vector by a time
ratio (total time/observed time) to obtain the total, average distance
for all vectors in that quadrant. Then, we converted the relative
distances into µm and divided this total, extrapolated distance by the
total gastrulation time (25.2 minutes) to obtain the average rate of
movement. Angle/direction of movement was calculated by
calculating the arctangent of (y/x).
Blastomere recombination experiments
P1 cells were isolated and allowed to develop in EGM. To rotate partial
isolates relative to each other, P1 isolates were first split at one of three
cell boundaries. For Ea and Ep recombinations, Ea and Ep were
separated ~5 minutes after division and recombined. For MSxx and
P4 recombinations, the MS or P3 cell was separated from the rest of
the isolate and recombined one cell division later, when E had divided
into Ea and Ep. Although the partial P1 isolates were not deliberately
rotated, we refer to them as rotated because they were placed back in
contact without regard for their original rotational orientations;
examining centrosome positions has demonstrated that this procedure
can randomize rotational orientations (Goldstein, 1995). The
recombined P1 isolates were mounted on coverslips and filmed as
described above. Analysis of the direction of movement depended on
whether MSxx and P4 moved in the same plane or not. In all cases,
MSxx and P4 always moved towards the Ea/Ep boundary. If both cells
moved in the same plane, the movement was either 0° (towards each
other) or 180° (away from each other). If a cell moved in or out of
plane, a reference cell was chosen (see Fig. 8) and the non-reference
cell was estimated to move 45°, 90° or 135° off the axis of movement
of the reference cell.
RESULTS
Gastrulation can occur in vitro
In C. elegans, gastrulation is initiated at the 26-cell stage by
the ingression of the endoderm precursor cells, Ea and Ep
(Sulston et al., 1983). As Ea and Ep leave the eggshell, a
space appears between the E cells and the eggshell (Fig.
1B,C). Six wedge-shaped, neighboring cells converge over
the ventral surface of the E cells: P4, three granddaughters of
MS (MSpp, MSpa, MSap), ABplpa and ABplpp (Fig. 1D).
From a lateral view, P4, the germline precursor cell, is
adjacent to Ep on the posterior side, while MSxx, one
granddaughter of the mesodermal precursor MS cell, is
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J.-Y. Lee and B. Goldstein
Fig. 1. C. elegans gastrulation. (A) Early embryonic lineage of C. elegans development. Daughters are annotated ‘a’ for anterior, ‘p’ for
posterior, ‘l’ for left, and ‘r’ for right. Anterior is towards the left. The endoderm precursors, Ea and Ep, are labeled in blue. The neighboring
cells that close the gastrulation cleft are in green (ABplpa, ABplpp, MSap, MSpa, MSpp and P4). The scale on the left refers to time, in
minutes, since fertilization. (B) Confocal images of lateral view of gastrulating embryos labeled with the membrane marker SynaptoRed to
better visualize cell boundaries. Ea and Ep ingress towards the center of the embryo, and are eventually surrounded by MSap and P4. Asterisks
indicate Ea and Ep and neighboring cells are labeled with arrows in B-D. (C,D) Panels show DIC time-lapse views of gastrulation. (C) Lateral
view of gastrulation, similar to B. Images are at 10 minute intervals. (D) Ventral view of gastrulation. From left to right, time intervals are 0, 12
and 32 minutes. As Ea and Ep ‘sink’ into the embryo, six cells close up the ventral cleft. Note that while ABplpa and ABplpp start moving
toward the cleft, they divide and the posterior daughters of these cells finish the movement. In this and all figures, embryos are oriented anterior
towards the left, and posterior towards the right. Scale bars: 10 µm. Movies of time-lapse images are available online at
http://dev.biologists.org/supplemental/.
adjacent to Ea on the anterior. P4 and MSxx can be seen
moving toward each other into the space left by the E cells,
and they meet on the ventral side of Ea and Ep (Fig. 1B,C).
Therefore, the ingression of Ea and Ep involves a total of
eight cells, almost a third of the cells in the embryo at this
stage. After this initial internalization of Ea and Ep, P4 and
the MS descendents ingress ~1 hour later, and gastrulation
continues as additional cells ingress through the 300-cell
stage (Nance and Priess, 2002). This internalization of Ea and
Ep places the presumptive endoderm in the center of the
embryo, as occurs in gastrulation in other organisms (Keller
et al., 1991).
The C. elegans embryo develops inside a vitelline envelope,
which is surrounded by a chitinous eggshell (Wood, 1988). It
has previously been reported that gastrulation does not occur
in devitellinized embryos, suggesting that the vitelline
envelope might produce a micro-environment required for
gastrulation (Schierenberg and Junkersdorf, 1992). However,
half of the embryos cultured in that study did not make
rhabditin/gut granules, a marker of endoderm differentiation in
C. elegans embryos, suggesting that the embryonic culture
medium used might not have supported development as well
as EGM (see Materials and Methods).
To determine if gastrulation can occur in vitro, we
devitellinized embryos, filmed their development in EGM, and
compared them with intact embryos (Fig. 2A-H). Gastrulation
movements occurred consistently in devitellinized embryos
(Fig. 2E-H, six out of six embryos), and these movements
occurred at the same time as they do in intact embryos (see
below). Therefore, the eggshell and the vitelline envelope do
not serve as required surfaces for gastrulation forces to act
against or as sources for signals that are necessary for
gastrulation to occur.
Gastrulation does not require AB descendants
Gastrulation movements in the descendents of isolated P1 cells
were previously noted in some partial embryos (Laufer et al.,
1980; Edgar, 1995). We wanted to extend what was observed
by asking if these movements occur consistently in vitro, and
to what extent the movements were similar to those seen in
intact embryos. First, we tested whether gastrulation requires
AB descendants, either as substrates for cell crawling or as a
source of essential signals.
Time-lapse imaging of P1 isolates showed that gastrulation
movements occur consistently in vitro without AB descendants
(Fig. 2I-P, 12/12 embryos). Without the eggshell or the AB
descendants as reference points, we could not assess whether
or not the E cells in the P1 isolates were moving into what was
once the center of the embryo. However, we always observed
P4 and MSxx moving towards each other, as they do in intact
embryos (12/12 embryos), although not to the same extent (see
below). The descendants of P1 exhibited variable division
patterns, producing partial embryos that ranged from a dumbbell shape, in which the neighbors of the E cells made multiple
cell-cell contacts with the E cells (Fig. 2I-L), to a linear
orientation, in which there were single cell-cell contacts
between the E cells and their neighbors (Fig. 2M-P). Partial
embryos of all geometries underwent gastrulation movements
(Fig. 2I-P). As we observed that both the leading and trailing
edges of the neighboring cells were displaced in the direction
of movement (data not shown), we infer that these are directed
movements rather than cells simply spreading over the surfaces
of Ea and Ep. Our results suggest that single cell-cell contacts
between the E cells and their immediate neighbors, P4 and
MSxx, are sufficient for at least some gastrulation movements
to occur.
Because P1 isolates would enable us to perform cell
Cell positioning in C. elegans
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Fig. 2. Gastrulation in intact embryos,
devitellinized embryos and P1 isolates.
Asterisks indicate Ea and Ep, arrows
indicate MSxx and P4. Time-lapse
images were time-standardized
between all four sets, with 0 minutes
indicating the start of gastrulation
movements. (A-D) Time-lapse images
of gastrulation in an intact embryo
(same embryo as Fig. 1C), (E-H) in a
devitellinized embryo, and (I-P) in P1
isolates. Variation of starting
orientation between intact and
devitellinized embryos is due to
devitellinization (A versus E). The two
sets of P1 isolate images represent two
different division patterns, either in a
dumb-bell orientation (I-L) or in a
linear orientation (M-P). Linear
orientation occurs in less than 10% of
all P1 isolates (data not shown). Other
division patterns were variations
between these two extremes. Scale
bars: 10 µm. Movies of time-lapse
images available online at
http://dev.biologists.org/supplemental/.
manipulations that are not feasible in intact, or even
devitellinized, embryos, we were interested in determining to
what extent gastrulation movements occurred normally in
descendents of P1 isolates. Thus, we next asked to what degree
the movements we observed were similar to those in intact
embryos in both the timing as well as the extent (distance) of
the movements. In intact embryos, gastrulation always began
after Ea and Ep were born, but before they divided (Fig. 3A,
10/10 embryos). Similarly, the onset of movements in both
devitellinized embryos (six out of six embryos) and in P1
isolates (12/12 embryos) always occurred before the end of the
Ea/Ep cell cycle (Fig. 3A).
Next, we quantified the extent of movement by measuring
the angle of movement of P4 and MSxx relative to a reference
axis defined by the positions of Ea and Ep nuclei (see Materials
and Methods). As a negative control, we chose mom-2 mutants,
as gastrulation fails in embryos produced by mom-2 mutant
mothers (Rocheleau et al., 1997; Thorpe et al., 1997). mom-2
mutants, like all gastrulation-defective mutants identified to
date in C. elegans (Knight and Wood, 1998; Shi and Mello,
1998; Zhu et al., 1997; Tabara et al., 1999), have shortened Ea
and Ep cell cycles, with Ea and Ep dividing about 15 minutes
earlier than wild type. Therefore, as a time reference, we used
the cell cycles of neighboring cells because those are not
different in wild-type and mom-2 embryos.
In wild-type, intact embryos, gastrulation movements can
begin as early as P4 birth (Fig. 3A). Because our measurements
are affected by cell divisions, and because MSxx is born 12
minutes after P4, we first examined movement of P4 during this
12 minute interval before MSxx birth. The amount of P4 cell
movement from all three wild-type groups (intact embryos,
devitellinized embryos and P1 isolates) was significantly
greater than that of P4 movement in mom-2 mutant embryos
(Fig. 3B, Table 1).
Next, we examined the angle of movement by both P4 and
MSxx during the 30 minute period from when MSxx is born
until it divides. MSxx moved significantly more in each of the
wild-type groups than in P1 isolates from mom-2 mutant
embryos (Fig. 3C, Table 1). Additionally, MSxx movement in
another gastrulation-defective mutant, gad-1 (Knight and
Wood, 1998), was much less than in wild-type groups (data not
shown). We were surprised to find that P4 cells, during this time
period, moved to a similar extent in both wild-type P1 isolates
and P1 isolates derived from mom-2 embryos. This result is not
specific to mom-2, as analysis of gad-1 produced a similar
result (data not shown). The data suggests that the P4 cells in
these mutants may retain the ability to move or be translocated
during this time period, or that the extent of P4 movement in
wild-type P1 isolates is not as robust as that of MSxx
movement.
We conclude that devitellinized embryos and P1 isolates
exhibit several aspects of gastrulation movements seen in
intact embryos. First, the onset of movements occurs at the
same time in intact embryos, devitellinized embryos, and in
P1 isolates. Second, the movements of P4 and MSxx are
always in the same direction as in intact embryos.
Additionally, we found that in most cases, the extent of cell
movement in wild-type P1 isolates was significantly greater
than that of gastrulation-defective mutant P1 isolates. We note,
however, that the extent of some cell movements in intact
embryos is significantly greater than that in devitellinized
embryos and in P1 isolates (Table 1), suggesting that the
eggshell, vitelline envelope and/or AB cells may be required
for the full extent of gastrulation movements.
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J.-Y. Lee and B. Goldstein
A
B
Ep
Ea
90
P4
0o
o
WT
Intact
WT
Devit
27.6 o
14.5
o
0o
Ea, Ep
born
WT
P1
mom-2
P1
15.7 o
3.2 o
P4 born
C
MSxx
0o
Ep
Ea
P4
MSxx
born
52.0o
WT
Intact
WT
Devit
o
25.9
P4
WT
P1
Ea, Ep
divide
19.8
o
mom-2
P1
14.2 o
56.5o
WT
Devit
WT
Intact
Intact Embryos (n=10)
Devitellinized Embryos (n=6)
P1 Isolates (n=12)
33.1 o
MSxx
40.9 o
WT
P1
mom-2
P1
8.2 o
Fig. 3. Timing and extent of gastrulation movements. (A) Timing of the beginning of gastrulation movements in intact embryos (light gray
circles), devitellinized embryos (medium gray circles) and P1 isolates (black circles) with respect to the length of the Ea and Ep cell cycle. The
timing of gastrulation was normalized between embryos (see Materials and Methods). Black ticks along Ea/Ep lineage represent a fifth of the
total cell cycle. The early lineage is shown for reference. (B,C) Quantification of P4 and MSxx movement relative to Ea and Ep in wild-type
(WT) intact, devitellinized, P1 isolates and mom-2 P1 isolates. The data are shown by quadrant graphs, with gray unbroken lines representing
individual cases and black broken lines representing the mean for all embryos. A diagram representing the measurement method is shown
above the graphs. (B) Quantification of P4 movement relative to Ea and Ep from the time P4 is born until MSxx is born (12 minutes).
(C) Quantification of MSxx and P4 movement relative to Ea and Ep after MSxx is born (30 minutes).
MSxx does not chemotax towards P4
It is possible that P4 and MSxx send chemotactic signals that
could be used as cues to move towards each other. We tested
this hypothesis by removing the potential sources of
chemoattractants. Either MS or P2 (the grandparents of MSxx
and P4) were removed from P1 isolates, and the truncated
isolates were subsequently filmed to document cell movements
in the manipulated embryos. When we removed P2, MSxx
appeared to move as it would in unmanipulated P1 isolates (Fig.
4D-F). Analysis with angle measurements confirmed this
observation, as the average extent of movement of MSxx in
truncated isolates was similar to that found in normal P1
isolates (Fig. 4H) and was significantly greater than the
movement seen in mom-2 P1 isolates (Table 1). Although we
also observed movement by P4 after removing MS, the
movement was not significantly greater than that in
gastrulation-defective mutants, or significantly less than that in
wild-type P1 isolates (Table 1). Therefore, we cannot make a
conclusion for P4 on the basis of this experiment alone. We
conclude that MSxx does not rely on a chemotactic signal from
P4 during gastrulation.
Intact microfilaments are required for gastrulation
As the cytoskeleton plays a central role in cell motility, we
asked whether microtubules and/or microfilaments were
required for gastrulation. To test this, we permeabilized intact,
gastrulating embryos by laser ablating holes in the eggshell and
then exposed the embryos to various cytoskeletal inhibitors.
Cell positioning in C. elegans
313
Fig. 4. MSxx and P4 do not
chemotax towards each other
during gastrulation. Asterisks
indicate Ea and Ep, an ‘X’
indicates the removed cells,
and the remaining neighbor
is labeled with an arrow.
(A-C) P4 moves with respect
to Ea and Ep in the absence
of MS. (D-F) MSxx moves
with respect to Ea and Ep in
the absence of P4. (G) Angle
quantification of six sets of
each manipulation, as in Fig. 3. The angle measurements for P4 represent only the first 12 minutes of movement, as this number was significant
from mom-2 P4 (see Results, Table 1). Scale bars: 10 µm.
Table 1. Angle measurements in intact embryos, devitellinized embryos and P1 isolates
Average movement (degrees, ±s.d.)
Genotype
Wild-type (N2)
mom-2(or139)
Wild-type (N2)
Manipulation
Intact
Devitellinized
P1 isolate
P1 isolate
EMS (no P2)
E+P2 (no MS)
P4 (first 12 minutes)*
(±16.9)‡
(±7.5)‡
(±5.7)‡
27.6
14.5
15.7
3.17 (±1.8)
NA
9.83 (± 4.9)‡
P4 (30 minutes)†
(±20.4)§
(±10.9)¶,**
(±10.9)¶,**
52.0
25.9
19.8
14.17 (±4.7)
NA
19.9 (± 12.0)¶
MSxx (30 minutes)
(±3.9)††
(±13.4)††,**
(±24.1)††
56.5
33.1
40.9
8.16 (±3.4)
25.9 (±14.0)††
NA
n
6
6
6
6
6
7
*The time period between P4 birth and MSxx birth.
†The time of MSxx birth until MSxx division. For the range of numbers, see Fig. 3B and Fig. 4G,H.
Differences between wild-type groups in each time period were not significantly different from each other, except in cases indicated by **, where differences
were seen between devitellinized embryos or P1 isolates compared to intact embryos. No significant differences were seen between devitellinized embryos and P1
isolates.
n, number of cases; NA, not applicable.
‡P<0.025 versus mom-2 P isolate P , first 12 minutes.
1
4
§P<0.05 versus mom-2 P isolate P , 30 minutes.
1
4
¶P>0.05 versus mom-2 P isolate P , 30 minutes.
1
4
††P<0.025 versus mom-2 P isolate MSxx.
1
Exposing the embryos to taxol, which suppresses microtubule
dynamics in living cells (Yvon et al., 1999), or to the
microtubule-destabilizing drug nocodazole, had the expected
effects on microtubule distribution in gastrulating embryos, but
neither drug prevented gastrulation (Fig. 5A-C). We conclude
that microtubules do not play an essential role specifically
during C. elegans gastrulation in producing movements.
By contrast, all embryos subjected to the microfilament
assembly inhibitors cytochalasin D or latrunculin B did not
gastrulate (Fig. 5E,F), suggesting that intact microfilaments
are required for gastrulation. Examination of the actin
distribution following drug treatment confirmed that actin
microfilaments were no longer cortically enriched following
treatment (Fig. 5E,F). To determine whether microfilaments
are required only for gastrulation to initiate, or also for cells
to continue moving, we exposed embryos to cytochalasin D
during the middle of gastrulation, when MSxx and P4 were
about halfway towards meeting each other. Gastrulation
halted immediately upon exposure to the drug (six out of six
embryos). To ensure that cytochalasin D was not simply
killing the embryos, we performed washout experiments
to test the reversibility of the drug. Upon washout of
cytochalasin D, gastrulation consistently resumed, after an
average of 11±6 minutes (15/15 embryos). In addition, when
we examined the rate of movement, we confirmed that during
cytochalasin D treatment, the E cells did not ingress (Table 2).
After washout, the rate of Ea/Ep movement was roughly
equivalent to untreated control embryos (Table 2). Therefore,
intact microfilaments are required throughout C. elegans
gastrulation.
Cells do not appear to move via a classical crawling
mechanism
Actin-dependent mechanisms of motility mediated by cell
protrusions are well documented in other motility systems,
such as growth cones, keratocytes and fibroblasts (Mitchison
and Cramer, 1996). We were interested in whether gastrulating
cells in C. elegans also move by a similar mechanism.
Crawling cells typically send out actin-rich protrusions, in the
form of filopodia or lamellipodia, towards the direction of
movement. Actin polymerization at the leading edge of the
cell, in combination with traction at the cell base, results in
forward displacement of the cell body. Simultaneously, the
cortex and cell surface move rearward relative to the direction
of movement. Finally, the very rear of the cell must de-adhere
from the substrate so that there is a total forward motion of the
314
J.-Y. Lee and B. Goldstein
Fig. 5. Microfilaments are required for
gastrulation. Asterisks indicate Ea and Ep.
Confocal imaging of live embryos
documents the qualitative, not quantitative,
effects of pharmacological agents on
tubulin and actin distribution. (A-C) Effects
of drugs on tubulin in live embryos
expressing Tubulin::GFP strain, treated with
(A) 1% DMSO (control), (B) 50 µM taxol
and (C) 10 µM nocodazole. (D-F) Effects
of drugs on actin in live embryos expressing
Actin::YFP, treated with (D) 1% DMSO,
(E) 5 µg/ml cytochalasin D and (F) 10 µM
latrunculin B. 1% DMSO treatment was the
control as this was the final concentration of
DMSO used for the drug studies.
Gastrulation progressed normally in the
presence of 1% DMSO. To the right of each
image are the numbers of embryos assayed
for gastrulation. The embryos imaged were
different from those used for the assay.
Scale bar: 10 µm.
Table 2. Rate of Ea/Ep ingression following cytochalasin D
treatment
Average movement (µm/minute±s.d.)
Treatment
Control
Cytochalasin D
During drug
exposure
After washout/during
gastrulation
n
NA
–0.095 (±0.082)
0.227 (±0.067)
0.281 (±0.055)
3
3
Rates were calculated from kymographs (see Materials and Methods). The
time window measured started at the ingression of Ea and Ep and ended with
their division. Positive numbers indicate movement in the direction of the
center of the embryo. Slower movement in the opposite direction occurs in
cytochalasin D as cells lose their shape.
cell (Mitchison and Cramer, 1996). In the case of gastrulation,
Ea and Ep could be actively migrating towards the center of
the embryo, or the neighboring cells could be crawling towards
each other, using Ea and Ep as a substrate.
We used several independent methods to test if cells were
indeed crawling via actin-mediated protrusions. Our initial
approach was to look for extensions or cell shape changes in
gastrulating P1 isolates. Tracings of the cell shapes from timelapse images during the time of gastrulation showed that P4 and
MSxx exhibited subtle cell shape changes at the leading edge
towards the direction of movement, while Ea and Ep did not
make any such observable shape changes (data not shown).
This suggested that P4 and MSxx might be making extensions
and crawling towards each other, using the E cells as substrates.
To test this, we used scanning electron microscopy (SEM) to
investigate whether or not the cells produced extensions.
Examination of both devitellinized embryos and P1 isolates
by SEM showed no consistent evidence of filopodia or
lamellipodia (intact, n=8, data not shown; P1 isolates, n=9, Fig.
6A). In some cases (four out of eight intact embryos, two out
of nine P1 isolates), we saw flattened protrusions at cell
boundaries reminiscent of those reported previously in
devitellinized embryos (Nance and Priess, 2002). However,
there was never more than one cell per embryo exhibiting a
protrusion and the cell that exhibited the protrusion varied from
embryo to embryo. In another approach, we reasoned that if
the cells had actin-rich extensions, then we should see an
increased intensity of filamentous actin (F-actin) at the leading
edge of the motile cells. Staining P1 isolates with fluorescent
phalloidin, which marks F-actin, did not reveal an enrichment
of actin at any specific cell-cell boundary (n=8, Fig. 6B).
Another marker of active protrusions is phosphotyrosine,
which has been shown to be enriched at the leading edge of
crawling cells including nematode sperm and Aplysia growth
cones (Italiano et al., 1996; Wu and Goldberg, 1993). We
observed no polarized enrichment of phosphotyrosine at any
specific cell-cell leading edge (n=12, Fig. 6C, and legend). We
also used PAR-2::GFP as a live cortical marker in P4 (Boyd et
al., 1996) and found that the cortex of the P4 cell did not make
protrusions as it moved over the ventral surface of Ep (data not
shown). Finally, tracking the surfaces of cells with fluorescent
markers showed that the surface of MSxx does not move
rearwards relative to the movement of the cell body, as would
be expected from protrusion based crawling (see below).
Together, these approaches suggest that cells do not form
lamellipodia or filopodia and are unlikely to move via
protrusions during C. elegans gastrulation.
Myosin is required for cell movement
If cells do not move via protrusion-based cell crawling, then
they must either move through another microfilamentdependent process, or microfilaments must play an indirect role
in movement. Recent evidence showed an accumulation of
NMY-2, a non-muscle myosin type II, at the ventral cortex of
Ea and Ep during gastrulation (Nance and Priess, 2002), but
whether or not this accumulation was required for gastrulation
was not tested. Therefore, we asked if myosin activity was
required for gastrulation.
As loss of NMY-2 results in severe defects in establishment
of polarity and in execution of cytokinesis very early in
embryogenesis, before gastrulation (Guo and Kemphues, 1996),
we tested the role of myosin activity using ML-7, a potent and
specific inhibitor of myosin light chain kinase (Saitoh et al.,
1987). As ML-7 has not been used previously in C. elegans
Cell positioning in C. elegans
315
Gastrulation-Inhibited (%)
120
100
80
60
40
ML-7
ML-9
H-7
20
0
0
1000
2000
3000
4000
5000
6000
Inhibitor Concentration (Micromolar)
Fig. 6. No lamellipodia or filopodia form in P1 isolates. Ea and Ep
are labeled with asterisks, and arrows indicate presumptive leading
edges of MSxx and P4. (A) Scanning electron micrograph of a P1
isolate. (B) F-actin localization. (C) Phosphotyrosine localization.
An enrichment of phosphotyrosine was seen at the Ep/P4 boundary,
but is unlikely to be functionally significant as it was not polarized in
the direction of movement and it was not present at the Ea/MSxx
boundary. Additionally, it was recently shown that phosphotyrosine
accumulates at the EMS/P2 boundary due to a signaling pathway that
is not implicated in gastrulation (Bei et al., 2002). Scale bars: 10 µm.
embryos, we performed dose response experiments to test the
specificity of the effect of ML-7 and two related compounds,
ML-9 and H-7, on gastrulation. ML-9 is also a MLCK inhibitor,
but has ten-times less affinity for MLCK compared with the
affinity of ML-7, whereas H-7 is a specific inhibitor of PKA, but
exhibits a very weak affinity for myosin light chain kinase
(Saitoh et al., 1987; Mabuchi and Takano-Ohmuro, 1990). We
found that while 250 µM ML-7 was sufficient to completely
inhibit gastrulation, 750 µM ML-9 and 4 mM H-7 were required
to inhibit gastrulation (Fig. 7). We also found that exposure of
embryos to 250 µM ML-7 immediately halted cell movement at
the beginning of gastrulation (six out of six embryos), as well as
in the middle of gastrulation (seven out of seven embryos). 750
µM H-7 was insufficient to inhibit gastrulation, but these
embryos arrested early, before morphogenesis, suggesting other,
potentially PKA-dependent defects (seven out of seven
embryos). These experiments suggest that ML-7 specifically
inhibits MLCK in C. elegans embryos and that myosin activity
is required for cell movements throughout C. elegans
gastrulation.
Cell surface tracking shows evidence of cell
contraction
Our experiments with pharmacological agents suggest that
actin and myosin play an essential role in C. elegans
Fig. 7. ML-7 and ML-9, myosin light chain kinase inhibitors, are
more potent inhibitors of gastrulation than H-7, a PKA inhibitor.
Embryos were assayed as gastrulation-inhibited if the ventral
surfaces of Ea and Ep were not fully covered by P4 and MSxx by 1
hour after exposure to inhibitor. Ki (inhibition coefficient, expressed
in µM) of the drugs against MLCK are as follows: ML-7, 0.3; ML-9,
3.8; H-7, 97 (Mabuchi and Takano-Ohmuro, 1990). We found that
the doses required to completely inhibit gastrulation were as follows:
ML-7, 250 µM; ML-9, 750 µM; H-7, 4 mM. We note that as much as
300 µM was required to inhibit MLCK in Xenopus growth cones
(Ruchhoeft and Harris, 1997), and high concentrations may be
required to out-compete the reservoir of endogenous ATP, as both
ML-7 and ML-9 are ATP analogs (Saitoh et al., 1987). Each data
point represents between four and 13 embryos.
gastrulation, but the mechanism of movement is still unclear.
One possible mechanism is that MSxx and P4 could be actively
moving via a rotating mechanism, where the cells would roll
like wheels towards each other onto the ventral sides of Ea and
Ep, possibly mediated by local gradients in cell adhesion. An
alternative model is that a ventral, actomyosin-based
contraction toward the Ea and Ep border drives the neighboring
cells closer together. One prediction of this model is that the
apical (ventral) surfaces of Ea and Ep should decrease in size
during gastrulation, while embryos treated with the myosin
inhibitor would not. We therefore measured the distance along
the apical surface between the Ea/MSx(x) border and the Ep/P4
border (see Materials and Methods). We found that untreated,
control embryos underwent an average apical reduction of 7.7
(±2.3) µm during gastrulation, whereas the apical surfaces of
embryos treated with ML-7 was reduced by 1.1 (±2.3) µm
during the same time frame. Therefore, the apical surface areas
of Ea and Ep decrease during gastrulation, while myosininhibited embryos do not undergo significant changes.
Although these measurements are consistent with the
contraction model, they are also consistent with models in
which cells roll and also with models where the apical surfaces
decrease as a result of basolateral expansion. To assess directly
how cell surfaces behave, we tracked the surfaces of cells with
fluorescent microspheres and filmed them during gastrulation.
Fluorescent microspheres are useful markers for tracing the
movement at the cell surface because they do not become
engulfed by cells and are relatively photostable (Wang et al.,
1994). Thus, if Ea and Ep were undergoing actomyosin
contraction, then the microspheres on the ventral cortex of Ea
and Ep would converge toward the Ea/Ep boundary. However,
if the neighboring cells were rotating, then the microspheres
316
J.-Y. Lee and B. Goldstein
Fig. 8. Microsphere-marking of cell
surfaces reveals that the ventral surfaces
of Ea and Ep contract during
gastrulation. (A) Kymograph derived
from movements of microspheres
(white) during gastrulation from one film
of a wild-type P1 isolate. In the
kymograph, the image in each frame of
the time-lapse recording is used to
generate horizontal lines of image data
that are pasted together in descending
order; hence, time is represented on the
y-axis. Each pixel in a horizontal line of
image data was created by selecting the
brightest pixel in a 20-pixel high region.
As a result, horizontal movement of
microspheres can be seen as horizontal
movement of white dots as the
kymograph proceeds from top to bottom.
First frame and last frame used for the
kymograph are above and below the
kymograph, respectively. Ea and Ep are
labeled with asterisks, the Ea/Ep
boundary is marked by yellow
arrowhead in still frames and by the yellow line in the kymograph, and the white arrow indicates the
direction of MSxx movement. The microspheres on Ep can be seen converging towards each other
during gastrulation movements. (B) Summary of microsphere movements traced from all ten
simultaneous DIC/GFP films. Each arrow indicates the total displacement and angle of movement
by each microsphere. ‘X’ indicates microspheres that did not exhibit any displacement relative to the
displacement of the cell. (C) Average of the vectors (see Materials and Methods). Insets show the
directions of microsphere movement from each quadrant, with the average direction in gray. The
average velocity for each set of vectors is shown below the box.
on the neighboring cell surfaces would be displaced in the
direction of movement. Finally, if Ea and Ep were basolaterally
expanding, then the beads on the apical surface of Ea and Ep
should either remain stationary or move slightly away from the
Ea/Ep boundary.
Partial embryos were incubated with microspheres, and a
large number of microspheres were observed on the ventral
surfaces of Ea, Ep and MSxx; whether or not this distribution
is nonrandom and reflective of an increased degree of
adhesivity at these surfaces is unclear. As these surfaces had a
sufficiently large number of microspheres, we limited our
analysis to these surfaces. We found that microspheres on the
ventral sides of Ea and Ep generally moved toward the Ea-Ep
boundary (Fig. 8). We also observed microspheres on the
ventral side of MSxx moving along the cell surface towards Ea
(Fig. 8). These results are consistent with the model that the
ventral sides of Ea and Ep contract, and that MSxx might
additionally contribute to movement by rotating towards Ea
during gastrulation.
Polarity in Ea and Ep dictates the direction of
movement of neighboring cells
The observations that P4 and MSxx move toward each other in
P1 isolates, that a non-muscle myosin is enriched in the ventral
side of Ea and Ep (Nance and Priess, 2002), that myosin
activity is required for gastrulation and that the ventral sides of
Ea and Ep contract during gastrulation suggest that Ea and Ep
may be ingressing via a myosin-based contraction of their
ventral surfaces. One prediction of this hypothesis is that Ea
and Ep should be capable of dictating the direction of
movement of their neighbors, regardless of the orientation of
the neighbors. To test this, we rotated cells relative to each
other by bisecting a P1 isolate at various cell boundaries,
recombining the isolated halves, and filming the partial
embryos to document the ensuing direction of movement (see
Materials and Methods, Fig. 9). We tested three variations of
rotation experiments, in which we separated and recombined
P1 isolates at the Ea and Ep, Ea and MSxx, or Ep and P4
boundaries.
We found that when we separated and recombined Ea and
Ep, MSxx and P4 did not move towards each other in the same
axis as they did in unmanipulated P1 isolates. Instead, the cells
moved toward the Ea/Ep boundary in a range of directions
relative to each other (Fig. 9A). Thus, when Ea/Ep orientation
was randomized, so was the direction of movement by MSxx
and P4. In addition, because the neighbors move in different
directions, this result further confirms that MSxx and P4 are
not chemotaxing towards each other.
In isolates where MSxx and Ea were separated and
recombined, MSxx always moved in the same direction as P4
(10/10 embryos; Fig. 9B). Similarly, in most cases, P4 moved
in the same direction as MSxx where P4 had been recombined
with Ep (seven out of ten embryos; Fig. 9C). Curiously, in a
small number of Ep/P4 recombination cases, the neighboring
cells moved in the same plane toward each other, but in the
opposite direction (three out of ten embryos; Fig. 9C). It is
possible that P4 may influence the orientation by which polarity
is established in Ep, although this has not been tested. We
Cell positioning in C. elegans
Experimental Schematic
317
Result
A
B
C
Fig. 9. Ea and Ep polarity directs the movement of their neighbors.
On the left side of each panel is a schematic drawing of part of a P1
isolate and the experiment performed. Cells in the isolates were
separated at the site indicated by the broken line, rotated along an
axis (unbroken gray line in A), and recombined in their normal
orientations. The arrow below the cells indicates the direction of cell
movement by the reference cell. On the right of each panel is the
corresponding side view, in which the P1 isolates are oriented as if
looking down from one end, either P4 or MSxx (as indicated). Yellow
arrows indicate the direction of movement of P4 cells and the green
arrows indicate the direction of movement of MSxx cells. Separation
and recombination of (A) Ea and Ep, with P4 as the reference cell;
(B) MSxx and Ea, with P4 as the reference cell; or (C) P4 and Ep,
with MSxx as the reference cell.
conclude that Ea and Ep are polarized in a way that can
generally dictate the direction of movement of their neighbors.
DISCUSSION
Mechanism of movement in C. elegans gastrulation
Our results suggest a mechanism for cell movement during
C. elegans gastrulation, whereby an actomyosin-based
contraction constricts the ventral surfaces of the endodermal
precursor cells, pulling neighboring cells underneath and
resulting in internalization of the future endoderm (Fig. 10).
As all of the neighboring cells, regardless of their orientations,
move towards the ventral surface of the ingressing cells (Fig.
9), and as beads placed on the ingressing cells will move in
this direction (Fig. 8), we speculate that ventral contraction of
Fig. 10. Model of cell contraction during C. elegans gastrulation.
(Top) Ea and Ep with myosin enriched at the ventral side. Hatching
represents myosin, while gray arc underneath the cells represents the
eggshell. Myosin-based contraction causes the ventral side of Ea and
Ep to constrict, bringing neighbors closer to each other, which
pushes Ea and Ep into the center of the embryo (bottom).
the ingressing cells is sufficient to move the neighboring cells.
Given that enrichments of NMY-2 have been observed at the
ventral side of other cells that ingress later in gastrulation
(Nance and Priess, 2002), it is tempting to speculate that this
same mechanism is used repeatedly to internalize cells in the
C. elegans embryo.
Additional mechanisms may contribute additively or
redundantly to ventral contraction of Ea and Ep. There are
indications that some neighboring cells may make an active
contribution to movement. For example, in P1 isolates, MSxx
sometimes moves not only to the Ea/Ep boundary but also
beyond this point (data not shown), and beads on the ventral
surface of MSxx move in the direction of cell movement (Fig.
8), suggesting that MSxx might be rolling in this direction,
perhaps driven by cell-cell adhesion. This does not appear to
occur in P4, as P4 never passes the Ea/Ep boundary in P1
isolates (data not shown).
Although many mechanisms may contribute to C. elegans
gastrulation, other potentially redundant mechanisms can be
excluded by our results. First, our cell manipulation
experiments suggest that neighboring cells do not chemotax
towards each other. Second, the cells most likely do not move
by protrusion-based cell crawling, as no protrusive structures
were observed during gastrulation, and surfaces do not move
in the directions expected in protrusion-based cell crawling.
Thus, C. elegans gastrulation could be used as a model to
examine cell motility that is not dependent on cell protrusions.
Additionally, because the descendants of P1 isolates can
gastrulate in vitro, we conclude that at least three classes
of mechanisms for cell positioning are not essential for
gastrulation movements, although the possibility that they
make redundant or additive contributions to movements cannot
be discounted. First, buckling forces or spatial restriction of
cell divisions requiring physical constraints, such as those
provided by the eggshell and vitelline envelope, cannot be
318
J.-Y. Lee and B. Goldstein
necessary for gastrulation because gastrulation can occur in
devitellinized embryos. For example, the P3 division in intact
embryos is aligned with the eggshell, so that when P4 is born,
it might push Ep towards the center of the embryo. However,
oriented P3 cell division constrained by the eggshell cannot
be required to initiate gastrulation movements because
gastrulation can occur in vitro.
Second, we can rule out the necessity of a neighbor
annealing mechanism in which, as the neighboring cells (P4,
MSpp, MSpa, MSap, ABplpa and ABplpp) converge on the
ventral side of Ea and Ep (Fig. 1C), their adhesiveness to each
other drives their convergence, sealing up the ventral cleft. As
the AB cells are not required for gastrulation movements, and
as movements can occur in P1 isolate orientations in which the
only neighboring cells, P4 and MSxx, do not contact each other
until the end of gastrulation movements (Fig. 2M-P), we
conclude that neighbor annealing is not required for
gastrulation.
Third, a model whereby movement is driven exclusively by
differential adhesion between cells is unlikely. The differential
adhesion hypothesis (Steinberg, 1963) proposes that cell
rearrangements can be directed by differences in the adhesive
strengths of cells. Cells that show some degree of motility tend
to maximize adhesive contacts, resulting in the most adhesive
cells ending up in the center of a group of cells, with the less
adhesive cells surrounding them (Steinberg and Takeichi,
1994). For example, it is possible that Ea and Ep could be
more adhesive than their neighbors, and this differential
adhesion could drive their ingression as they become
surrounded by their less adhesive neighbors. However,
differential adhesion alone cannot be a sufficient mechanism
as Ea and Ep in linear P1 isolates exhibit gastrulation
movements without being surrounded by neighboring cells. In
addition, there is, to date, no evidence for a role for adhesion
molecules in C. elegans gastrulation, as molecules such as
catenins, cadherins and extracellular matrix components do
not show upregulation in the E cells at the time of gastrulation,
and embryos carrying mutations in genes that encode adhesion
proteins do not arrest until late embryogenesis, during the
events of ventral closure and elongation (reviewed by
Michaux et al., 2001). However, large-scale knockouts of
adhesion molecules have not been performed, and functional
redundancy may have prevented a gastrulation phenotype
from being detected thus far.
Cell contraction in morphogenesis
Our model of ventral contraction (Fig. 10) is reminiscent of
apical constriction, a long-proposed mechanism for certain
morphogenetic events. Cells that contract at their apical sides
basolaterally expand, which in a cell sheet can cause bending
or invagination of the sheet at the site of the apically
constricting cells (Lewis, 1947; Odell et al., 1981). Because of
their shape, these cells are often described as flask or bottle
cells (Rhumbler, 1899; Rhumbler, 1902; Ruffini, 1925). Apical
constriction has been suggested to play a role in morphogenetic
movements in a wide range of organisms, including
gastrulation in shrimp (Hertzler and Clark, 1992), fly (Young
et al., 1991), jellyfish (Byrum, 2001), sea urchin (Kimberly and
Hardin, 1998), white sturgeon (Bolker, 1993), rabbit (Viebahn
et al., 1995) and frog (Keller, 1981; Hardin and Keller, 1988),
as well as in primitive streak formation in chick and rat
embryos (Solursh and Revel, 1978), and neurulation in frogs
(Jacobson et al., 1986). Whether apical constriction actually
drives shape changes in cell sheets has been directly tested in
Xenopus, sea urchin and Drosophila gastrulation. In Xenopus
and sea urchin embryos, removal or ablation of bottle cells
showed that the initial invagination of the epithelial sheet
required the bottle cells (Keller, 1981; Hardin and Keller, 1988;
Kimberly and Hardin, 1998). The cellular mechanism by which
bottle cells invaginate is unclear. In Drosophila embryos,
myosin and actin are enriched at the apical cortex of
gastrulating cells (Young et al., 1991). In addition, disrupting
a pathway that regulates actin through RhoA prevents
concerted apical constriction, halting gastrulation (Barrett et
al., 1997; Hacker and Perrimon, 1998). However, some clusters
of cells still undergo apical constriction, suggesting that this
pathway is required for concerted cell shape changes, but not
for apical constriction.
The apical constriction model predicts a local actomyosindriven cell contraction (Odell et al., 1981). Our results
demonstrate that C. elegans gastrulation is an example of apical
constriction. We define the apical and basal sides of cells based
on findings by Nance and Priess (Nance and Priess, 2002) that
cell cortices facing the outside of the embryo, or apical side,
have an enrichment of the protein PAR-3. NMY-2 also
accumulates at the apical side (Nance and Priess, 2002) and we
show, through pharmacological studies, that both actin and
myosin activity are required for gastrulation. Furthermore, the
apical surfaces of Ea and Ep contract, and the polarity of Ea
and Ep is important for the direction of movement by their
neighbors. As adherens junctions and tight junctions are not
found this early in embryogenesis (Krieg et al., 1978), we
conclude that apical constriction can function to position
blastomeres in early embryos, even before such cell-cell
junctions form. The availability of both genetic tools and direct
manipulation of cells should contribute to the usefulness of C.
elegans as a system for understanding the mechanism of apical
constriction in early embryos.
We thank Bruce Bowerman, Karen Oegema, Tony Hyman,
Christian Malone and Ken Kemphues for strains; Sharon Milgram
and Ted Salmon for antibodies and reagents; Victoria Madden,
Robert Bagnell and Tony Perdue for SEM and confocal assistance;
Jeff Molk, Julie Canman, Dale Beach, Rebecca Cheeks and Bill
Mohler for imaging assistance and advice; and Kerry Bloom and
his laboratory for generous use of their microscope setup. Some
strains used in this work were provided by the Caenorhabditis
Genetics Center, which is funded by the National Center for
Research Resources of the NIH. J. L. thanks Nathan Hall for
statistical analysis and moral support. We especially thank Mark
Peifer, Jean-Claude Labbé, Mythreye Karthikeyan and Nate Dudley
for critical reading of the manuscript, as well as members of the
Goldstein laboratory and Ray Keller for helpful discussions. This
work was supported by a March of Dimes Basil O’Connor Starter
Scholar Award to B.G, who is a Pew Scholar in the Biomedical
Sciences.
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